A large number of human cancers display genetic alterations that aberrantly activate the G1-control kinases, cyclin D-associated Cdk4 and Cdk6, suggesting critical roles for these proteins in oncogenic transformation. Escape from senescence is requisite for transformation. Senescence induced by Ras activation or continuous in vitro proliferation is accompanied by increased expression of p16ink4a and ARF. p16ink4a inhibits Cdk4 and Cdk6, leading to enhance growth-inhibitory action of Rb. ARF stabilizes p53, resulting in induction of another Cdk inhibitor, p21Cip1/Waf1. These senescence-associated pathways are critical for tumor suppression. Cdk4-null mouse embryonic fibroblasts (MEF) proliferate normally, but are resistant to oncogenic transformation upon Ras activation with p53 inhibition or Ink4a/ARF disruption. Senescence without ARF-p53 function accounts for this lack of transformation potential. Furthermore, Cdk4-null mice display reduced susceptibility to carcinogen- or radiation-induced tumorigenesis. Thus, Cdk4 disruption may effectively render cells insensitive to transformation, by inducing senescence under conditions that normally immortalize cells. The long-term goal is to establish the basis for therapeutic intervention of oncogenic transformation. This proposal will evaluate the hypothesis that aberrant activation of cyclin D/Cdk4 and cyclin D/Cdk6 is required for a cell to overcome the senescence-dependent tumor suppressive mechanism, leading to immortalization and tumorigenesis. The specific aims are: (1) Determine how Cdk4 disruption leads MEF to senescence under inhibition of the ARF-p53 pathway, by investigating senescence/immortalization-associated proteins in Cdk4-null MEF; (2) Determine whether Cdk4 is required for Ras-induced carcinogenesis of the mammary epithelium in mice, by examining MMTV-Ras transgenic mice with mammary-specific Cdk4 disruption; (3) Determine how Cdk4 and Cdk6 interact in immortalization, transformation and oncogenesis, by examining Cdk6-null MEF and mice. These studies are expected to clarify essential roles of the cyclin D-dependent kinases during oncogenesis. This program takes innovative approaches that should lead us to better understanding of the oncogenic interplay of Cdk4 and Cdk6 with the senescence-dependent tumor suppressive pathways, using unique mouse and cell models.